The hormone erythropoietin (Epo), which interacts with a high affinity cell surface receptor on the erythropoietin receptor (EpoR), developing erythroblasts is critical for normal erythroid development. Intracellular signaling consequent to binding of the ligand to its receptor provides a proliferative response and ensures differentiation of the erythroid lineage. We have recently cloned the genomic DNA of the human EpoR. In its 5' flanking region, there are potential regulatory sequences specific to the erythroid cell lineage. We have studied the tissue specificity of the expression of the hEpoR gene using transient transfection reporter gene assays and have also determined the extent that GATA-1, a transcription factor with erythroid specificity, can transactivate the hEpoR promoter. We used transient transfection assays to examine the activity of the hEpoR promoter in human erythroleukemia cell lines, OCIM1 and K562, which express high and low levels of EpoR protein on the cell surface, respectively. Deletion analysis of the 5' flanking DNA of the hEpoR promoter linked to a luciferase reporter gene and transfected into OCIM1 and K562 cells indicate that much of the transcription activity is contained within a 150 bp proximal promoter. In HeLa cells, which do not express EpoR, transfected EpoR promoter was transactivated by cotransfection with a GATA-1 expression vector. Cotransfection with GATA-1 was also carried out in the erythroleukemia cells with constitutive levels of GATA-1. We observe that increasing GATA-1 levels in K562 cells can further increase EpoR promoter activity by 6 fold. In contrast, no significant increase was observed in OCIM1 cells in which the endogeneous EpoR gene is already active. The levels of EpoR mRNA in OCIM1 cells is greater than in K562 cells. These results suggest that GATA-1 levels in OCIM1 may be saturating, and increasing GATA-1 expression has little effect on EpoR promoter activity and explain the dependency of EpoR expression which parallels the increase and decrease in GATA-1 levels during differentiation and maturation of erythroid progenitors. At the level of transcriptional activation, the interactions of GATA-1 or other transcriptional factors with the EpoR promoter should improve understanding of erythroid specific gene regulation.